Methods and compositions for treating polycystic ovary syndrome

ABSTRACT

The present invention relates to methods of treating polycystic ovary syndrome (PCOS) comprising administering glucagon-like peptide-1 (GLP-1), exendin, and analogs and agonists thereof, to subjects suffering therefrom.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/317,126 filed Dec. 11, 2002, and is a continuation-in-part ofInternational Application No. PCT/US03/01109, which is a continuation ofU.S. application Ser. No. 10/317,126 filed Dec. 11, 2002 and claimspriority to U.S. Provisional Application No. 60/350,395 filed Jan. 22,2002.

FIELD OF THE INVENTION

The present invention relates to endocrinology and pharmacology. Moreparticularly, it relates to methods and compositions for treatingsubjects suffering from polycystic ovary syndrome (PCOS).

BACKGROUND OF THE INVENTION

Polycystic ovary syndrome (PCOS), also known as polycystic ovariandisease or Stein-Leventhal syndrome, affects an estimated 6-10% of womenin the United States. PCOS is characterized by anovulation (irregular orabsent menstrual periods) and hyperandrogenism (elevated serumtestosterone and androstenedione). Additional etiological and clinicalsymptoms of this disease can include abnormal uterine bleeding, enlargedmultifollicular ovaries, infertility, obesity, insulin resistance,hyperinsulinemia, hypertension, hyperlipidemia, type-2 diabetesmellitus, excess facial hair growth, hair loss and acne.

Insulin resistance and hyperinsulinemia are highly prevalent in patientswith PCOS and are thought to underlie the pathophysiology of thisdisease (Udoff, L., et al., Curr. Opin. Obstret. Gynecol. 7:340-343(1995); Barbieri, R. L., Am. J Obstet. Gynecol. 183:1412-8 (2000); Kim,L. H. et al., Fertility and Sterility 73:1097-1098 (2000); Iuorno, M. J.et al., Obstet. Gynecol. Clin. North Am. 28:153-164 (2001); Zacur, H.Z., Obstet. Gynecol. Clin. North Am. 28:21-33 (2001)). Recent studiessuggest that the hyperandrogenism associated with PCOS is caused by anincrease in ovarian androgen production (e.g., testosterone andandrostenedione) and a decrease in serum androgen-binding globulinconcentration, due to hyperinsulinemia. Insulin has been shown todirectly stimulate production of androgens by the ovary, at least inpart by increasing the activity of P450c17α, an enzyme involved in theproduction of testosterone in the ovarian theca cells (Iuorno, M. J. etal., supra). At the level of the pituitary axis, hyperandrogenismsuppresses follicle stimulating hormone (FSH) secretion, altersgonadotropin-releasing hormone (GnRH) release and increases lutenizinghormone (LH) secretion. These abnormalities, along with the localeffects of androgens on the ovaries, lead to follicular involution,anovulation, and infertility. Similarly, oligomenorrhea and amenorrheaoccur and are interspersed with heavy vaginal bleeding. Hyperinsulinemiamay also lead to high blood pressure and increased clot formation andhas been implicated in the development of cardiovascular disease, strokeand type-2 diabetes (Iuorno, M. J. et al., supra; Zacur, H. A., supra).

Traditionally, treatment of PCOS was directed primarily at correctingthe underlying symptoms. For example, hirsutism and menstrualirregularities were treated with anti-androgenic drugs, including birthcontrol pills, spironolactone, flutamide or finasteride. Infertilitytreatments have included weight loss diets, ovulation medications (e.g.,clomiphene, follistim and Gonal-F), so-called “ovarian drilling”surgery, and in vitro fertilization. More recent treatments for PCOS aretargeted towards lowering insulin levels. Insulin-sensitizing agentssuch as metformin, D-Chiro-inositol, diazoxide, and PPAR-gammainhibitors (e.g., troglitazone (Rezulin), rosiglitazone (Avandia) andpioglitazone (Actos)), have been demonstrated to restore fertility andreverse the endocrine abnormalities associated with PCOS. Althoughmetformin and PPAR-gamma inhibitors do not interfere with pregnancy,they are generally discontinued during pregnancy because of concern overtheir safety and effect(s) on the fetus. Moreover, women with PCOS whobecome pregnant experience spontaneous abortion during the firsttrimester at rates as high as 30% to 50% (Iuorno, M. J. et al., supra;Zacur, H. A., supra; Phipps, W. R., Obstet. Gynecol. Clin. North Am.28:165-182 (2001). Thus, there is a need for new and better compositionsand methods for treating PCOS.

SUMMARY OF THE INVENTION

Applicants have solved the above problem by discovering thatglucagon-like peptide-1 (GLP-1), exendin, and agonists and analogs ofthese compounds are capable of lowering insulin resistance or increasinginsulin sensitivity. The present invention relates to methods fortreating PCOS using GLP-1, GLP-1 agonists, exendin, or exendin agonists.In one embodiment, the methods of this invention comprise administeringto a patient a therapeutically effective amount of GLP-1, GLP-1agonists, exendin, or exendin agonists. In another embodiment, themethod comprises reducing or preventing insulin resistance in a subjectsuffering from PCOS. In yet another embodiment, the method comprisespreventing the onset of type-2 diabetes in a subject suffering fromPCOS. In a further embodiment, the method comprises restoring regularmenses, ovulation, or fertility in a subject suffering from PCOS.

In a preferred embodiment, the GLP-1 agonist is a GLP-1 analog withagonist activity, and the exendin agonist is an exendin analog withagonist activity. In another preferred embodiment, the subject is human.

By GLP-1 agonist is meant a compound that mimics the effects of GLP-1 onPCOS by binding to the receptor or receptors where GLP-1 causes thiseffect. Certain GLP-1 analogs with agonist activity are described inChen et al., U.S. Pat. No. 5,512,549, issued Apr. 30, 1996, entitledGlucagon-Like Insulinotropic Peptide Analogs, Compositions and Methodsof Use. Other GLP-1 analogs with agonist activity are described inJohnson et al., U.S. Pat. No. 5,574,008, issued Nov. 12, 1996, entitled,Biologically Active Fragments of Glucagon-Like Insulinotropic Peptide.Still other GLP-1 analogs with agonist activity are described in Buckleyet al., U.S. Pat. No. 5,545,618, issued Aug. 13, 1996, entitled GLP-1Analogs Useful for Diabetes Treatment. All three referenced U.S. patentsare incorporated herein by this reference.

By an exendin agonist is meant a compound that mimics the effects ofexendin on PCOS by binding to the receptor or receptors where exendincauses this effect. Certain exendin analogs having agonist activity aredescribed in U.S. Provisional Patent Application Ser. No. 60/055,404,filed Aug. 8, 1997, which enjoys common ownership with the presentinvention and is hereby incorporated by this reference. Certain otherexendin analogs with agonist activity are described in U.S. ProvisionalPatent 2 5 Application Ser. Nos. 60/066,029 and 60/065,442, both filedNov. 14, 1997 which enjoy common ownership with the present inventionand are hereby incorporated by this reference. Preferred exendin analogshaving agonist activity include those described in U.S. ProvisionalPatent Application Ser. Nos. 60/055,404 and 60/065,442.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including the definitions, will control. Allpublications, patents and other references mentioned herein areincorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, suitablemethods and materials are described below. The materials, methods, andexamples are for illustrative purposes only, and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

In order to further define the invention, the following terms anddefinitions are herein provided.

The term “alopecia” as used herein, refers to a condition in which apatient experiences loss of hair due to, e.g., infections of the scalpor skin, nervousness, or a specific disease such as PCOS. The hair mayfall out in patches or there may be diffuse loss of hair instead ofcomplete baldness in one area.

The term “exendin” refers to naturally occurring exendin peptides thatare found in the salivary secretions of the Gila-monster. Preferredexendins include exendin-3 [SEQ. ID. NO. 7], which is present in thesalivary secretions of Heloderma harridum, and exendin-4 [SEQ. ID. NO.9], which is a peptide present in the salivary secretions of Helodermasuspectum (Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng.,J., et al., J. Biol. Chem., 267:7402-05, 1992). Exendin-4, as it occursin the salivary secretions of the Gila monster, is an amidated peptide.However, it should be understood that the term “exendin,” “exendin-3,”and “exendin-4” refer to both the amidated form of the peptide and theacid form of the peptide.

Exendin-4 was first thought to be a (potentially toxic) component of thevenom. It now appears that exendin-4 is devoid of toxicity, and that itinstead is made in salivary glands in the Gila monster. The exendinshave some sequence similarity to several members of the glucagon-likepeptide family, with the highest homology, 53%, being to GLP-1[7-36]NH2(Goke, et al., J. Biol. Chem., 268:19650-55, 1993). “Exendin agonist”refers to compounds that mimic the effect of an exendin by binding tothe receptor or receptors where the exendin causes this effect. Exendin“agonist activity” in this context means having the biological activityof an exendin, but it is understood that the activity of the analog canbe either less potent or more potent than the native exendin. Otherexendin agonists include, e.g., chemical compounds specifically designedto active that receptor or receptors at which an exendin exerts itsaffect on PCOS.

The terms “GLP-1,” and “glucagon-like peptide-1,” according to thisinvention both refer to GLP-1 amide [SEQ. ID. NO. 4], as well as theacid form of GLP-1, which is also known as proglucagon[78-107]. “GLP-1agonist” refers to compounds that mimic the effects of GLP-1 on PCOS bybinding to the receptor or receptors where GLP-1 causes this effect.Preferred GLP-1 agonists are GLP-1 analogs with agonist activity. GLP-1“agonist activity” in this context means having the biological activityof GLP-1 (7-36) amide (GLP-1(7-36)NH₂), but it is understood that theactivity of the analog can be either less potent or more potent thannative GLP-1(7-36) amide. Other GLP-1 agonists include, e.g., chemicalcompounds specifically designed to activate the receptor or receptors atwhich GLP-1 exerts its affect on PCOS.

The term “hirsutism” as used herein, refers to a condition in which apatient exhibits abnormal hairiness.

The term “hyperandrogenism” as used herein, refers to a condition inwhich a patient exhibits elevated levels of androgens (e.g.,testosterone, androstenedione) in serum.

The term “hyperinsulinemia” as used herein, refers to a condition inwhich a patient exhibits elevated plasma insulin levels.

The term “hyperlipidemia” as used herein, refers to a condition in whicha patient exhibits elevated concentrations of any or all lipids inplasma.

The term “hypertension” as used herein, refers to a condition in which apatient experiences persistently high blood pressure (i.e., a systolicpressure equal or greater than 140 mm Hg and a diastolic pressure equalto or greater than 90 mm Hg).

The term “insulinotropic” as used herein, refers to an ability tostimulate the release of insulin into the circulation.

The term “insulin resistance” as used herein, describes a subnormalbiological response to a given concentration of insulin (i.e., decreasedglucose transport across the cell membrane in response to insulin).

The term “pharmaceutically acceptable carrier or adjuvant” as usedherein, refers to a non-toxic carrier or adjuvant that may beadministered to a patient together with a compound of the invention, andwhich does not destroy the pharmacological activity thereof.

The terms “polycystic ovarian syndrome,” “PCOS,” “polycystic ovariandisease” or “Stein-Leventhal syndrome,” as used herein refer to adisease affecting women. Women diagnosed with PCOS may exhibit one ormore of the following symptoms: anovulation (irregular or absentmenstrual periods), hyperandrogenism (elevated serum testosterone and/orandrostenedione), abnormal uterine bleeding, enlarged multifollicularovaries, infertility, obesity, insulin resistance, hyperinsulinemia,hypertension, hyperlipidemia, type-2 diabetes mellitus, excess facialhair growth, hair loss, and acne.

The terms “therapeutically or pharmaceutically effective” or“therapeutically or pharmaceutically effective amount” refers to anamount of the compound of this invention required to reduce or lessenthe severity of PCOS or any of its symptoms (e.g., of insulinresistance, hyperinsulinemia, type-2 diabetes mellitus, obesity,hypertension, hyperlipidemia, anovulation or irregular ovulation,infertility, hyperandrogenism, hirsutism, alopecia, acne, enlargedmultifollicular ovaries and abnormal uterine bleeding, for some periodof time). A therapeutically or pharmaceutically effective amount alsomeans the amount required to improve the clinical symptoms of a patient.

The term “type-2 diabetes mellitus” as used herein, refers to a disease,also known as non-insulin-dependent diabetes mellitus (NIDDM) oradult-onset diabetes mellitus (AODM), in which a patient has elevatedconcentrations of blood sugar levels.

The present invention relates to methods for treating PCOS in a patient.The methods include administering to a subject a therapeuticallyeffective amount of GLP-1, exendin, and agonists and analogs of thesecompounds. The methods of this invention further relate to loweringinsulin resistance using GLP-1, exendin, and agonists and analogs ofthese compounds. Many of the symptoms associated with PCOS stem from anunderlying insulin resistance.

Glucagon-Like Peptide-1 (GLP-1)

GLP-1 plays a key role in the regulation of plasma glucose homeostasis.It is involved in stimulating insulin secretion and inhibiting glucagonrelease by the pancreas, inhibiting gastric acid secretion and motility,and suppressing appetite and food intake. GLP-1 is a member of theincretin group of secretagogue hormones that are released fromintestinal enteroendocrine cells in response to the ingestion of food.GLP-1 binds to the GLP-1 receptor, which is expressed on the β-cells ofthe pancreas. Binding of GLP-1 to its receptor triggers an intracellularsignaling pathway that results in stimulation of insulin secretion withconcomitant inhibition of glucagon production. This in turn leads toinhibition of hepatic glucose production, which lowers blood glucoselevels. Although the role of GLP-1 in maintaining plasma glucoseconcentration is well established, prior to this invention, it was notknown that GLP-1 is also capable of increasing insulin sensitivity.

Mammalian GLP peptides and glucagon are encoded by the same gene. In theileum, the precursor is processed into two major classes of GLP peptidehormones, namely GLP-1 and GLP-2. GLP-1(1-37) has the sequence:His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly[SEQ ID NO: 1]. GLP-1(1-37) is amidated post-translationally to yieldGLP-1(1-36)NH₂, which has the sequence:His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(NH₂)[SEQ ID NO: 2], or is enzymatically processed to yield GLP-1(7-37),which has the sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly[SEQ ID NO: 3]. GLP-1(7-37) can also be amidated to yieldGLP-1(7-36)amide, which has the sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(NH₂)[SEQ ID NO: 4]. Likewise, GLP-1(1-36)amide can be processed toGLP-1(7-36)amide.

Intestinal L cells secrete GLP-1(7-37) and GLP-1(7-36)NH₂ in a ratio ofabout 1:5. These truncated forms of GLP-1 have short half-lives in vivo(less than 10 minutes), and are inactivated by an aminodipeptidase (DPPIV) to yield GLP-1(9-37), which has the sequence:Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly[SEQ ID NO: 5], and GLP-1(9-36)amide, which has the sequence:Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(NH₂)[SEQ ID NO: 6], respectively. It has been speculated that the peptidesGLP-1(9-37) and GLP-1(9-36) amide might affect hepatic glucoseproduction, but apparently they do not stimulate production or releaseof insulin from the pancreas.

As used in this specification, the term “GLP-1 analog” includesGLP-1(1-37), GLP-1-(1-36)NH₂, GLP-1(7-37), GLP-1(9-37), GLP-1(9-36)NH₂(“GLP-1(9-36) amide”). The present invention includes the use ofrecombinant human GLP-1 analogs and GLP-1 analogs derived from otherspecies, whether recombinant or synthetic.

As used in this specification, the term “GLP-1 agonist” includes anymolecules, whether they be peptides, peptide mimetics, or other chemicalcompounds, that bind to or activate a GLP-1 receptor or receptors atwhich GLP-1 exerts its affect on PCOS, such as the GLP-1(7-36)amidereceptor, and its second messenger cascade. For example, GLP-1 agonistsinclude molecules having insulinotropic activity and that are agonistsof (i.e., activate) the GLP-1 receptor molecule and its second messengeractivity on, inter alia, insulin producing β-cells.

“GLP-1 analogs” also include peptides that are encoded bypolynucleotides that express biologically active GLP-1 analogs withagonist activity, as defined herein. For instance, GLP-1 analogs may bepeptides containing one or more amino acid substitutions, additions ordeletions, compared with GLP-1(7-36)amide. In one embodiment, the numberof substitutions, deletions, or additions is 30 amino acids or less, 25amino acids or less, 20 amino acids or less, 15 amino acids or less, 10amino acids or less, 5 amino acids or less or any integer in betweenthese amounts. In one aspect of the invention, the substitutions includeone or more conservative substitutions. A “conservative” substitutiondenotes the replacement of an amino acid residue by another,biologically active similar residue. Examples of conservativesubstitutions include the substitution of one hydrophobic residue, suchas isoleucine, valine, leucine, or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, and the like. The following table lists illustrative, butnon-limiting, conservative amino acid substitutions. ORIGINAL RESIDUEEXEMPLARY SUBSTITUTIONS ALA SER, THR ARG LYS ASN HIS, SER ASP GLU, ASNCYS SER GLN ASN, HIS GLU ASP, GLU GLY ALA, SER HIS ASN, GLN ILE LEU,VAL, THR LEU ILE, VAL LYS ARG, GLN, GLU, THR MET LEU, ILE, VAL PHE LEU,TYR SER THR, ALA, ASN THR SER, ALA TRP ARG, SER TYR PHE VAL ILE, LEU,ALA PRO ALA

It is further understood that GLP-1 analogs include the above describedpeptides which have been chemically derivatized or altered, for example,peptides with non-natural amino acid residues (e.g., taurine, β- andγ-amino acid residues and D-amino acid residues), C-terminal functionalgroup modifications, such as amides, esters, and C-terminal ketonemodifications and N-terminal functional group modifications, such asacylated amines, Schiff bases, or cyclization, as found, for example, inthe amino acid pyroglutamic acid.

Also included in the present invention are peptide sequences havinggreater than 50% sequence identity, and preferably greater than 90%sequence identity to (1) SEQ ID NOS: 1, 2, 3, and 4; and (2) totruncated sequences thereof. As used herein, sequence identity refers toa comparison made between two molecules using standard algorithms wellknown in the art. The preferred algorithm for calculating sequenceidentity for the present invention is the Smith-Waterman algorithm,where SEQ ID NO: 1 [i.e., GLP-1(1-37)] is used as the reference sequenceto define the percentage identity of homologs over its length. Thechoice of parameter values for matches, mismatches, and insertions ordeletions is arbitrary, although some parameter values have been foundto yield more biologically realistic results than others. One preferredset of parameter values for the Smith-Waterman algorithm is set forth inthe “maximum similarity segments” approach, which uses values of 1 for amatched residue and −⅓ for a mismatched residue (a residue being eithera single nucleotide or single amino acid). Waterman, Bull. Math. Biol.46; 473 (1984). Insertions and deletions (indels), x, are weighted asx_(k)=1+⅓k, where k is the number of residues in a given insert ordeletion. Id.

For instance, a sequence that is identical to the 37-amino acid residuesequence of SEQ ID NO: 1, except for 18 amino acid substitutions and aninsertion of 3 amino acids, would have a percent identity given by:[(1×37 matches)−(⅓×18 mismatches)−(1+ 3/3 indels)]/37=78% “identity”

Agonists of glucagon-like peptide that exhibit activity through theGLP-1(7-36) amide receptor have been described. See EP 0708179 A2;Hjorth et al., J. Biol. Chem. 269; 30121 (1994); Siegel et al., Amer.Diabetes Assoc. 57^(th) Scientific Session, Boston (1997); Hareter etal., Amer. Diabetes Assoc. 57^(th) Scientific Session, Boston (1997);Adelhorst et al., J. Biol. Chem. 269, 6275 (1994); Deacon et al.,16^(th) International Diabetes Federation Congress Abstracts,Diabetologia Supplement (1997); Irwin et al., Proc. Natl. Acad. Sci. USA94; 7915 (1997); Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992).Göke & Byrne, Diabetic Medicine 13; 854 (1996). Recent publicationsdisclose Black Widow GLP-1 and Ser² GLP-1. See Holz & Hakner, Comp.Biochem. Physiol., Part B 121; 177 (1998) and Ritzel et al., J.Endocrinol 159; 93 (1998).

GLP-1 receptors are cell-surface proteins found, for example, oninsulin-producing pancreatic β-cells; the GLP-1(7-36) receptor has beencharacterised in the art. Methods of determining whether a chemical orpeptide binds to or activates a GLP-1 receptor are known to the skilledartisan.

The biological activity of a GLP-1 agonist can be determined by in vitroand in vivo animal models and human studies, as is well known to theskilled artisan. GLP-1 biological activity can be determined by standardmethods, in general, by receptor binding activity screening procedures,which involve providing appropriate cells that express the GLP-1receptor on their surface, for example, insulinoma cell lines such asRINmSF cells or INS-1 cells. See Mojsov, Int. J. Peptide Protein Res.40; 333 (1992) and EP 0708179 A2. Cells that are engineered to express aGLP-1 receptor also can be used. In addition to measuring specificbinding of tracer to membrane using radioimmunoassay methods, cAMPactivity or glucose dependent insulin production can also be measured.In one method, a polynucleotide encoding the GLP-1 receptor is employedto transfect cells so that they express the GLP-1 receptor protein.Thus, for example, these methods may be employed for screening for areceptor agonist by contacting such cells with compounds to be screenedand determining whether such compounds generate a signal (i.e., activatethe receptor). Other screening techniques include the use of cells thatexpress the GLP-1 receptor, for example, transfected CHO cells, in asystem to measure extracellular pH or ionic changes caused by receptoractivation. For example, potential agonists may be contacted with a cellthat expresses the GLP-1 protein receptor and a second messengerresponse (e.g., signal transduction or ionic or pH changes), may bemeasured to determine whether the potential agonist is effective.

Polyclonal and monoclonal antibodies can be utilized to detect, purify,and identify GLP-1-like peptides for use in the methods describedherein. Antibodies such as ABGA1178 detect intact GLP-1(1-37) orN-terminally-truncated GLP-1(7-37) or GLP-1(7-36) amide. Otherantibodies detect the end of the C-terminus of the precursor molecule, aprocedure that allows one—by subtraction—to calculate the amount ofbiologically active, truncated peptide (i.e., GLP-1(7-37)amide). Orskovet al., Diabetes 42; 658 (1993); Orskov et al., J. Clin. Invest. 1991,87; 415 (1991).

GLP-1 and the GLP-1 agonists of the invention that are peptides that canbe made by solid-state chemical peptide synthesis. Such peptides canalso be made by conventional recombinant techniques using standardprocedures described in, for example, Sambrook & Maniatis, MolecularCloning, A Laboratory Manual. “Recombinant,” as used herein, means thata gene is derived from a recombinant (e.g., microbial or mammalian)expression system that has been genetically modified to contain apolynucleotide encoding a GLP-1 peptide as described herein.

GLP-1 and the GLP-1 agonists of the invention that are peptides may be anaturally purified product, or a product of synthetic chemicalprocedures, or produced by recombinant techniques from prokaryotic oreukaryotic hosts (for example, by bacteria, yeast, higher plant, insect,or mammalian cells in culture or in vivo). Depending on the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention are generally non-glycosylated, but may beglycosylated. The GLP-1 peptides can be recovered and purified fromrecombinant cell cultures by methods including, but not limited to,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, and lectin chromatography.High-performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Exendins

Exendin-4, a 39-amino acid polypeptide, is a naturally occurring peptideisolated from the salivary secretions of the Gila monster. Animaltesting of exendin-4 has shown that its ability to lower blood glucosepersists for several hours. Exendin-4, as it occurs in the salivarysecretions of the Gila monster, is an amidated peptide. However, itshould be understood that the term “exendin,” and “exendin-4”specifically refers to both the amidated form of the peptide and theacid form of the peptide.

Certain exendin sequences are compared to the sequence of GLP-1 inTable 1. TABLE 1 a. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR(NH₂) b.HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH₂) c.DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH₂) d.HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH₂) e.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPSS f.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPS g.HSDAIFTEEYSKLLAKLALQKYLASILGSRTSPPP(NH₂) h.HSDAIFTQQYSKLLAKLALQKYLASILGSRTSPPP(NH₂)a = GLP-1(7-36) (NH₂) [SEQ. ID NO: 4].b = exendin 3 (NH₂) [SEQ. ID NO: 7].c = exendin 4 (9-39)(NH₂) [SEQ. ID NO: 8].d = exendin 4 (NH₂) [SEQ. ID NO: 9].e = helospectin I [SEQ. ID NO: 10].f = helospectin II [SEQ. ID NO: 11].g = helodermin (NH₂) [SEQ. ID NO: 12].h = Q⁸, Q⁹ helodermin (NH₂) [SEQ. ID NO: 13].

As described herein, the nonclinical pharmacology of exendin-4 has beenstudied. In the brain, exendin-4 binds principally to the area postremaand nucleus tractus solitarius region in the hindbrain and to thesubfornical organ in the forebrain. Exendin-4 binding has been observedin the rat and mouse brain and kidney. The structures to which exendin-4binds in the kidney are unknown.

Various experiments have compared the biologic actions of exendin-4 andGLP-1 and demonstrated a more favorable spectrum of properties forexendin-4. A single subcutaneous dose of exendin-4 lowered plasmaglucose in db/db (diabetic) and ob/ob (diabetic obese) mice by up to40%. In Diabetic Fatty Zucker (ZDF) rats, 5 weeks of treatment withexendin-4 lowered HbA_(1c) (a measure of glycosylated hemoglobin used toevaluate plasma glucose levels) by up to 41%. Insulin sensitivity wasalso improved by 76% following 5 weeks of treatment in obese ZDF rats.In glucose intolerant primates, dose-dependent decreases in plasmaglucose were also observed.

An insulinotropic action of exendin-4 has also been observed in rodents,improving insulin response to glucose by over 100% in non-fasted HarlanSprague Dawley (HSD) rats, and by up to ˜10-fold in non-fasted db/dbmice. Higher pretreatment plasma glucose concentrations were associatedwith greater glucose-lowering effects. Thus the observed glucoselowering effect of exendin-4 appears to be glucose-dependent, andminimal if animals are already euglycemic.

Exendin-4 dose dependently slowed gastric emptying in HSD rats and was˜90-fold more potent than GLP-1 for this action. Exendin-4 has also beenshown to reduce food intake in NIH/Sw (Swiss) mice following peripheraladministration, and was at least 1000 times more potent than GLP-1 forthis action. Exendin-4 reduced plasma glucagon concentrations byapproximately 40% in anesthetized ZDF rats during hyperinsulinemic,hyperglycemic clamp conditions, but did not affect plasma glucagonconcentrations during euglycemic conditions in normal rats. Exendin-4has been shown to dose-dependently reduce body weight in obese ZDF rats,while in lean ZDF rats, the observed decrease in body weight appears tobe transient.

The toxicology of exendin-4 has been investigated in single-dose studiesin mice, rats and monkeys, repeated-dose (up to 28 consecutive dailydoses) studies in rats and monkeys and in vitro tests for mutagenicityand chromosomal alterations. To date, no deaths have occurred, and therehave been no observed treatment-related changes in hematology, clinicalchemistry, or gross or microscopic tissue changes. Exendin-4 wasdemonstrated to be non-mutagenic, and did not cause chromosomalaberrations at the concentrations tested (up to 5000 μg/mL).

In support of the investigation of the nonclinical pharmacokinetics andmetabolism of exendin-4, a number of immunoassays have been developed. Aradioimmunoassay with limited sensitivity (˜100 pM) was used in initialpharmacokinetic studies. A two-site IRMA assay for exendin-4 wassubsequently validated with a lower limit of quantitation of 15 pM. Thebioavailability of exendin-4, given subcutaneously, was found to beapproximately 50-80% using the radioimmunoassay. This was similar tothat seen following intraperitoneal administration (48-60%). Peak plasmaconcentrations (C_(max)) occurred between 30 and 43 minutes (T_(max)).Both C_(max) and AUC values were monotonically related to dose. Theapparent terminal half-life for exendin-4 given subcutaneously wasapproximately 90-110 minutes. This was significantly longer than the14-41 minutes seen following intravenous dosing. Similar results wereobtained using the IRMA assay. Degradation studies with exendin-4compared to GLP-1 indicate that exendin-4 is relatively resistant todegradation.

As used in this specification, the term “exendin agonist” includes anymolecules, whether they be peptides, peptide mimetics, or other chemicalcompounds, that bind to or activate a receptor or receptors at whichexendin exerts its affect on PCOS, such as the GLP-1(7-36)amidereceptor, and its second messenger cascade. For example, exendinagonists include molecules having insulinotropic activity and that areagonists of (i.e., activate) the GLP-1 receptor molecule and its secondmessenger activity on, inter alia, insulin producing β-cells.

The structure activity relationship (SAR) of exendin was investigatedfor structures that may relate to the activity of exendin, for itsstability to metabolism, and for improvement of its physicalcharacteristics, especially as it pertains to peptide stability and toamenability to alternative delivery systems, and various exendin agonistpeptide compounds have been invented. Exendin agonists include exendinanalogs with agonist activity in which one or more naturally occurringamino acids are inserted, eliminated or replaced with another aminoacid(s). Preferred exendin analogs are peptide analogs of exendin-4.

Exendin analogs include peptides that are encoded by polynucleotidesthat express biologically active exendin analogs with agonist activity,as defined herein. For instance, exendin analogs may be peptidescontaining one or more amino acid substitutions, additions or deletions,compared with exendin-4. In one embodiment, the number of substitutions,deletions, or additions is 30 amino acids or less, 25 amino acids orless, 20 amino acids or less, 15 amino acids or less, 10 amino acids orless, 5 amino acids or less or any integer in between these amounts. Inone aspect of the invention, the substitutions include one or moreconservative substitutions. A “conservative” substitution denotes thereplacement of an amino acid residue by another, biologically activesimilar residue. Examples of conservative substitutions include thesubstitution of one hydrophobic residue, such as isoleucine, valine,leucine, or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, and the like.The following table lists illustrative, but non-limiting, conservativeamino acid substitutions. ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS ALASER, THR ARG LYS ASN HIS, SER ASP GLU, ASN CYS SER GLN ASN, HIS GLU ASP,GLU GLY ALA, SER HIS ASN, GLN ILE LEU, VAL, THR LEU ILE, VAL LYS ARG,GLN, GLU, THR MET LEU, ILE, VAL PHE LEU, TYR SER THR, ALA, ASN THR SER,ALA TRP ARG, SER TYR PHE VAL ILE, LEU, ALA PRO ALA

It is further understood that exendin analogs include the abovedescribed peptides which have been chemically derivatized or altered,for example, peptides with non-natural amino acid residues (e.g.,taurine, β- and γ-amino acid residues and D-amino acid residues),C-terminal functional group modifications, such as amides, esters, andC-terminal ketone modifications and N-terminal functional groupmodifications, such as acylated amines, Schiff bases, or cyclization, asfound, for example, in the amino acid pyroglutamic acid.

Also included in the present invention are peptide sequences havinggreater than 50% sequence identity, and preferably greater than 90%sequence identity to (1) SEQ ID NOS: 7 and 9; and (2) to truncatedsequences thereof. As used herein, sequence identity refers to acomparison made between two molecules using standard algorithms wellknown in the art. The preferred algorithm for calculating sequenceidentity for the present invention is the Smith-Waterman algorithm,where SEQ ID NO: 9 [i.e., exendin-4] is used as the reference sequenceto define the percentage identity of homologs over its length. Thechoice of parameter values for matches, mismatches, and insertions ordeletions is arbitrary, although some parameter values have been foundto yield more biologically realistic results than others. One preferredset of parameter values for the Smith-Waterman algorithm is set forth inthe “maximum similarity segments” approach, which uses values of 1 for amatched residue and −⅓ for a mismatched residue (a residue being eithera single nucleotide or single amino acid). Waterman, Bull. Math. Biol.46; 473 (1984). Insertions and deletions (indels), x, are weighted asx_(k)=1+⅓k, where k is the number of residues in a given insert ordeletion. Id.

Novel exendin analogs with agonist activity are described in commonlyowned PCT Application Serial No. PCT/US98/16387 filed Aug. 6, 1998,entitled “Novel Exendin Agonist Compounds,” which claims the benefit ofU.S. Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997, both ofwhich are herein incorporated by reference.

Other novel exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US98/24210, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds,” which claims thebenefit of U.S. Provisional Application No. 60/065,442 filed Nov. 14,1997, both of which are herein incorporated by reference.

Still other novel exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US98/24273, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds,” which claims thebenefit of U.S. Provisional Application No. 60/066,029 filed Nov. 14,1997, both of which are herein incorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US97/14199, filed Aug. 8,1997, entitled “Methods for Regulating Gastrointestinal Activity,” whichis a continuation-in-part of U.S. patent application Ser. No. 08/694,954filed Aug. 8, 1996, both of which are hereby incorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US98/00449, filed Jan. 7,1998, entitled “Use of Exendins and Agonists Thereof for the Reductionof Food Intake,” which claims priority to U.S. Provisional ApplicationNo. 60/034,90 filed Jan. 7, 1997, both of which are hereby incorporatedby reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US01/00719, filed Jan. 9,2001, entitled “Use of Exendins and Agonists Thereof for Modulation ofTriglyceride Levels and Treatment of Dyslipidemia,” which claimspriority to U.S. Provisional Application No. 60/175,365 filed Jan. 10,2000, both of which are hereby incorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US00/00902, filed Jan. 14,2000, entitled “Novel Exendin Agonist Formulations and Methods ofAdministration Thereof,” which claims priority to U.S. ProvisionalApplication No. 60/116,380 filed Jan. 14, 1999, both of which are herebyincorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US03/16699, filed May 28,2003, entitled “Novel Exendin Agonist Formulations and Methods ofAdministration Thereof,” which claims priority to U.S. application Ser.No. 10/157,224 filed May 28, 2002, both of which are hereby incorporatedby reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US00/00942, filed Jan. 14,2000, entitled “Methods of Glucagon Suppression,” which claims priorityto U.S. Provisional Application No. 60/132,017 filed Apr. 30, 1999, bothof which are hereby incorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US00/14231, filed May 23,2000, entitled “Use of Exendins and Agonists Thereof for the Treatmentof Gestational Diabetes Mellitus,” which claims priority to U.S.application Ser. No. 09/323,867 filed Jun. 1, 1999, both of which arehereby incorporated by reference.

Still other exendin analogs with agonist activity are described incommonly owned PCT Application Serial No. PCT/US99/02554, filed Feb. 5,1999, entitled “Inotropic and Diuretic Effects of Exendin and GLP-1,”which claims priority to U.S. Provisional Application No. 60/075,122filed Feb. 13, 1998, both of which are hereby incorporated by reference.

Activity as exendin agonists and exendin analogs with agonist activitycan be indicated, for example, by activity in the assays describedbelow. Effects of exendins or exendin agonists on PCOS can beidentified, evaluated, or screened for, using the methods describedherein, or other art-known or equivalent methods for determining effecton PCOS. Negative receptor assays or screens for exendin agonistcompounds or candidate exendin agonist compounds, such as a GLP-1receptor assay/screen described above, an amylin receptor assay/screenusing an amylin receptor preparation as described in U.S. Pat. No.5,264,372, issued Nov. 23, 1993, the contents of which are incorporatedherein by reference, one or more calcitonin receptor assays/screensusing, for example, T47D and MCF7 breast carcinoma cells, which containcalcium receptors coupled to the stimulation of adenyl cyclase activity,and/or a CGRP receptor assay/screen using, for example, SK-N-MC cells.

Certain preferred exendin analogs with agonist activity include:exendin-4 (1-30) [SEQ ID NO 14: His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Gly]; exendin-4 (1-30) amide [SEQ ID NO 15: His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile GluTrp Leu Lys Asn Gly Gly-NH₂]; exendin-4 (1-28) amide [SEQ ID NO 16: HisGly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn-NH₂]; ¹⁴Leu,²⁵Phe exendin-4 amide[SEQ ID NO 17: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln LeuGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro SerSer Gly Ala Pro Pro Pro Ser-NH₂]; ¹⁴Leu,²⁵Phe exendin-4 (1-28) amide[SEQ ID NO 18: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln LeuGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH₂]; and¹⁴Leu,²²Ala,²⁵Phe exendin-4 (1-28) amide [SEQ ID NO 19: His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu AlaIle Glu Phe Leu Lys Asn-NH₂].

Also included within the scope of the present invention arepharmaceutically acceptable salts of the compounds of formula (I-VIII)and pharmaceutical compositions including said compounds and saltsthereof.

Formula I

Exendin analogs with agonist activity also include those described inU.S. Provisional Application No. 60/065,442, including compounds of theformula (I) [SEQ ID NO. 20]: Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His, Arg or Tyr;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Ala, Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-   -   —NH₂    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,homoproline,        3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms.

Preferred exendin analogs include those wherein Xaa₁ is His or Tyr. Morepreferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycine orMet.

Preferred compounds are those wherein Xaa₂₅ is Trp or Phe.

Preferred compounds are those where Xaa₆ is Phe or naphthylalanine;Xaa₂₂ is Phe or naphthylalanine and

Xaa₂₃ is Ile or Val.

Preferred are compounds wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (I) whereinXaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ is Phe ornaphthylalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Phe ornaphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (I) wherein: Xaa₁ is His or Arg; Xaa₂is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₅ is Ala or Thr; Xaa₆ is Ala, Pheor nephthylalaine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ isAsp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala or Ser;Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu orpentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ SerSer-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Pro homoproline,thioproline or N-methylalanine; and Z₂ being —OH or —NH₂; provided thatno more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂,Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅,Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala. Especially preferred compounds includethose set forth in PCT application Serial No. PCT/US98/24210, filed Nov.13, 1998, entitled “Novel Exendin Agonist Compounds” identified thereinas compounds 2-23.

According to an especially preferred aspect, provided are compoundswhere Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds will be less susceptive tooxidative degration, both in vitro and in vivo, as well as duringsynthesis of the compound.

Formula II

Exendin analogs with agonist activity also include those described inU.S. Provisional Application No. 60/066,029, including compounds of theformula (II)[SEQ ID NO. 21]: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇, Ala Xaa₁₉ Xaa₂₀Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein:

Xaa₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala, Norval, Val, Norleu or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,homoproline,        3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or        N-alkylalanine; and

Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈, Xaa₉,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided alsothat, if Xaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ andXaa₉ is Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compoundsof formula (II) include those described in application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, identified therein in Examples 1-89 (“Compounds 1-89,”respectively), as well as those corresponding compounds identifiedtherein in Examples 104 and 105.

Preferred such exendin analogs include those wherein Xaa₁ is His, Ala orNorval. More preferably Xaa₁ is His or Ala. Most preferably Xaa₁ is His.

Preferred are those compounds of formula (II) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (II) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (II) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (II) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (II) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (II) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (II) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (II) include thosedescribed in application Serial No. PCT/US98/24273, filed Nov. 13, 1998,entitled “Novel Exendin Agonist Compounds” as having the amino acidsequence of SEQ. ID. NOS. 5-93 therein.

According to an especially preferred aspect, provided are compounds offormula (II) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula III

Also within the scope of the present invention are narrower genera ofcompounds having peptides of various lengths, for example genera ofcompounds which do not include peptides having a length of 28, 29 or 30amino acid residues, respectively. Additionally, the present inventionincludes narrower genera of compounds described in PCT applicationSerial No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” and having particular amino acid sequences, forexample, compounds of the formula (III) [SEQ. ID. NO. 22]: Xaa₁ Xaa₂Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇Xaa₂₈-Z₁;wherein:

Xaa₁ is His or Arg;

Xaa₂ is Gly or Ala;

Xaa₃ is Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu or pentylglycine;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;

Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, or Phe;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, thioproline and        N-methylylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and pharmaceuticallyacceptable salts thereof.

Formula IV

Additionally, the present invention includes narrower genera of peptidecompounds described in PCT Application Serial No. PCT/US98/24273, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as havingparticular amino acid sequences, for example, compounds of the formula[IV] [SEQ. ID. NO. 23]: Xaa₁ Xaa₂ Xaa₃ Xaa₅ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein:

Xaa₁ is His or Ala;

Xaa₂ is Gly or Ala;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Met or pentylglycine;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;

Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp or Phe;

Xaa₂₆ is Ala or Leu;

Xaa₂₇ is Ala or Lys;

Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Ser-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,homoproline,        thioproline, or N-methylylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇, and Xaa₂₈ are Ala; and provided that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla; and pharmaceutically acceptable salts thereof.

Preferred compounds of formula (IV) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (IV) include those wherein Xaa2 is Gly.

Preferred compounds of formula (IV) include those wherein Xaa4 is Ala.

Preferred compounds of formula (IV) include those wherein Xaa9 is Ala.

Preferred compounds of formula (IV) include those wherein Xaa14 is Leu,pentylglycine or Met.

Preferred compounds of formula (IV) include those wherein Xaa25 is Trpor Phe.

Preferred compounds of formula (IV) include those wherein Xaa6 is Ala,Phe or naphthylalanine; Xaa22 is Phe or naphthylalanine; and Xaa23 isIle or Val.

Preferred compounds of formula (IV) include those wherein Z1 is —NH2.

Preferred compounds of formula (IV) include those wherein Xaa31, Xaa36,Xaa37 and Xaa38 are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (IV) include those wherein Xaa39 is Seror Tyr, preferably Ser.

Preferred compounds of formula (IV) include those wherein Z2 is —NH2.

Preferred compounds of formula (IV) include those 42 wherein Z1 is —NH2.

Preferred compounds of formula (IV) include those wherein Xaa21 isLys-NH□-R where R is Lys, Arg, C1-C10 straight chain or branchedalkanoyl.

Preferred compounds of formula (IV) include those wherein X1 is Lys Asn,Lys-NHε-R Asn, or Lys-NHε-R Ala where R is Lys, Arg, C1-C10 straightchain or branched alkanoyl. Preferred compounds of formula (IV) includethose having an amino acid sequence described in PCT application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” as being selected from SEQ. ID. NOS. 95-110 therein.

Formula V

Also provided are compounds described in PCT application PCT/US98/24210,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”,including compounds of the formula (V) [SEQ. ID. NO. 24]: Xaa₁ Xaa₂ Xaa₃Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ X₁-Z₁;wherein

Xaa₁ is His, Arg or Tyr or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R, Lys-NH^(ε)—RAla, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, and Xaa₂₆ are Ala. Also within the scope of the presentinvention are pharmaceutically acceptable salts of the compound offormula (V) and pharmaceutical compositions including said compounds andsalts thereof.

Preferred exendin analogs of formula (V) include those wherein Xaa₁ isHis, Tyr or 4-imidazopropionyl. More preferably Xaa₁ is His.

Preferred are those compounds of formula (V) wherein Xaa₁ is4-imidazopropionyl.

Preferred are those compounds of formula (V) wherein Xaa₂ is Gly.

Preferred compounds of formula (V) are those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

According to one aspect, preferred are compounds of formula (V) whereinXaa₆ is Phe or naphthylalanine; and Xaa₂₂ is Phe or naphthylalanine; andXaa₂₃ is Ile or Val. More preferably, Z₁ is —NH₂. According to oneaspect, especially preferred are such compounds of formula (V) whereinXaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine. Morepreferrds, Z₂ is —NH₂.

Preferred compounds of formula (V) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl. Preferred compounds of formula (V)include compounds described in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and identified therein as Compound Nos. 62-69.

Preferred such exendin analogs include those wherein Xaa₁ is His, Ala orNorval. More preferably Xaa₁ is His or Ala. Most preferably Xaa₁ is His.

Preferred are those compounds of formula (V) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (V) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (V) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (V) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (V) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (V) wherein: Xaa₁ is His or Ala; Xaa₂is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ is Alaor Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ is Ala,Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine;Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ isAla, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala orGlu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg;Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Valor tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (V) include thosedescribed in PCT application Serial No. PCT/US98/24210, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds” and having the aminoacid sequences identified therein as SEQ. ID. NOS. 5-93.

According to an especially preferred aspect, provided are compounds offormula (V) where Xaa14 is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa25 is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula VI

Also provided are peptide compounds described in PCT Application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VI) [SEQ. ID. NO. 25]:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅Xaa₂₆ X₁-Z₁;wherein

Xaa₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Ala, Asp or Glu;

Xaa₄ is Ala, Norval, Val, Norleu or Gly;

Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser;

Xaa₈ is Ala, Ser or Thr;

Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser;

Xaa₁₂ is Ala or Lys;

Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu;

Xaa₁₆ is Ala or Glu;

Xaa₁₇ is Ala or Glu;

Xaa₁₉ is Ala or Val;

Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)-R where R is Lys, Arg, C¹⁻¹⁰ straightchain or branched alkanoyl or cycloalleyl-alkanoyl;

Xaa₂₂ is Phe, Tyr or naphthylalanine;

Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R, Lys-NH^(ε)—RAla, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;

provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈, Xaa₉,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, are Ala; and provided also that, if Xaa₁ isHis, Arg, Tyr, or 4-imidazopropionyl then at least one of Xaa₃, Xaa₄ andXaa₉ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VI) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (VI) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (VI) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (VI) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (VI) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VI) include those wherein Xaa₃₉ is Seror Tyr, preferably Ser.

Preferred compounds of formula (VI) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (VI) include those 42 wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₂₁ isLys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

Preferred compounds of formula (VI) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl.

Preferred compounds of formula (VI) include those described in PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” as having an amino acid sequenceselected from those identified therein as SEQ. ID. NOS. 95-110.

Formula VII

Compounds particularly useful according to the present invention areexendin analogs with agonist activity described in U.S. patentapplication Ser. No. 09/003,869, filed Jan. 7, 1998, entitled “Use ofExendins And Agonists Thereof For The Reduction of Food Intake”,including compounds of the formula (VII) [SEQ. ID. NO. 26]: Xaa₁ Xaa₂Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Ser Lys Gln Xaa₉ Glu Glu Glu AlaVal Arg Leu Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu Lys Asn Gly Gly Xaa₁₄ Ser SerGly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇, Xaa₁₈-Zwherein:

Xaa₁ is His, Arg or Tyr;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₄ is Phe, Tyr or naphthalanine;

Xaa₅ is Thr or Ser;

Xaa₆ is Ser or Thr;

Xaa₇ is Asp or Glu;

Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

Xaa₁₀ is Phe, Tyr or naphthalanine;

Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine;

Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline, 3Hyp,4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

with the proviso that the compound does not have the formula of eitherSEQ. ID. NOS. 7 or 9. Preferred N-alkyl groups for N-alkylglycine,N-alkylpentylglycine and N-alkylalanine include lower alkyl groupspreferably of 1 to about 6 carbon atoms, more preferably of 1 to 4carbon atoms. Also useful in the present invention are pharmaceuticallyacceptable salts of the compounds of formula (VII).

Preferred exendin analogs include those wherein Xaa₁ is His or Tyr. Morepreferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₉ is Leu, pentylglycine or Met.

Preferred compounds include those wherein Xaa₁₃ is Trp or Phe.

Also preferred are compounds where Xaa₄ is Phe or naphthalanine; Xaa₁₁is Ile or Val and Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

According to an especially preferred aspect, Xaa₁₅, Xaa₁₆ and Xaa₁₇ arethe same amino acid reside.

Preferred are compounds wherein Xaa₁₈ is Ser or Tyr, more preferablySer.

Preferably Z is —NH₂.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₄ isPhe or naphthalanine; Xaa₉ is Leu, pentylglycine or Met; Xaa₁₀ is Phe ornaphthalanine; Xaa₁₁ is Ile or Val; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₁₈ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (VII) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly; Xaa₃ is Asp or Glu; Xaa₄ is Phe or napthylalanine; Xaa₅ isThr or Ser; Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu orpentylglycine; Xaa₉ is Leu or pentylglycine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile, Val or t-butyltylglycine; Xaa₁₂ is Glu orAsp; Xaa₁₃ is Trp or Phe; Xaa₁₄, Xaa₁₅, Xaa₁₆, and Xaa₁₇ areindependently Pro, homoproline, thioproline, or N-methylalanine; Xaa₁₈is Ser or Tyr: and Z is —OH or —NH₂; with the proviso that the compounddoes not have the formula of either SEQ. ID. NOS. 7 or 9. Morepreferably Z is —NH₂.

According to an especially preferred aspect, provided are compoundswhere Xaa₉ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₁₃ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds are believed to exhibitadvantageous duration of action and to be less subject to oxidativedegration, both in vitro and in vivo, as well as during synthesis of thecompound.

Formula VIII

Also provided are compounds described in PCT Application Serial No.PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VIII) [SEQ. ID. NO. 27]:Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Ser Lys Gln Xaa₉ Glu GluGlu Ala Val Arg Leu Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu X₁ Gly Gly Xaa₁₄ Ser SerGly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Zwherein:

Xaa₁ is His, Arg, Tyr or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr;

Xaa₃ is Asp or Glu;

Xaa₄ is Phe, Tyr or naphthylalanine;

Xaa₅ is Thr or Ser;

Xaa₆ is Ser or Thr;

Xaa₇ is Asp or Glu;

Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

Xaa₁₀ is Phe, Tyr or naphthylalanine;

Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₁₂ is Glu or Asp;

Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine; X₁ is Lys Asn, Asn Lys,Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl;

Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline, 3Hyp,4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

with the proviso that the compound does not have the formula of eitherSEQ. ID. NOS. 7 or 9. Suitable compounds of formula (VIII) includecompounds described in PCT Application Serial No. PCT/US98/16387, filedAug. 6, 1998, entitled “Novel Exendin Agonist Compounds” having theamino acid sequences of SEQ. ID. NOS. 37-40 therein.

Preferred exendin analogs of formula (VIII) include those wherein Xaa₁is His, Tyr or 4-imidazopropionyl. More preferably, Xaa₁ is His or4-imidazopropionyl.

Preferred are those compounds of formula (VIII) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (VIII) wherein Xaa₉ is Leu,pentylglycine or Met.

Preferred are those compounds of formula (VIII) wherein Xaa₁₃ is Trp orPhe.

Preferred are those compounds of formula (VIII) wherein

X₁ is Lys Asn, or Lys-NH^(ε)—R Asn, where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl.

Also preferred are compounds of formula (VIII) wherein Xaa₄ is Phe ornaphthylalanine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile or Valand Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine. According to an especiallypreferred aspect, Xaa₁₈ is Ser or Tyr. Preferred are those suchcompounds wherein Xaa₁₈ is Ser. Preferably, Z is —NH₂.

According to one preferred aspect, preferred are compounds of formula(VIII) wherein Xaa₄ is Phe or naphthylalanine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile or Val, X₁ is Lys Asn, or Lys-NH^(ε)-RAsn, where R is Lys, Arg, C₁-C₁₀ straight chain or branched alkanoyl andXaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine.

In another embodiment, exendins and exendin analogs of the invention donot include the peptides of SEQ ID NOS. 7-13. In this embodiment,preferred exendin analogs include the analogs of Formulas (I-VIII), withthe proviso that the analogs do not include the peptides of SEQ ID NOs:7-13.

Exendins and exendin agonists that are peptides, such as exendinanalogs, described herein may be prepared through peptide purificationas described in, for example, Eng, et al., J. Biol. Chem. 265:20259-62,1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992, herebyincorporated by reference herein. Alternatively, exendins and exendinagonists that are peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al., J.Biol. Chem. 267:21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer. Thecompounds that constitute active ingredients of the formulations anddosages of the present invention may be prepared using standardsolid-phase peptide synthesis techniques and preferably an automated orsemiautomated peptide synthesizer. Typically, using such techniques, anα-N-carbamoyl protected amino acid and an amino acid attached to thegrowing peptide chain on a resin are coupled at room temperature in aninert solvent such as dimethylformamide, N-methylpyrrolidinone ormethylene chloride in the presence of coupling agents such asdicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of abase such as diisopropylethylamine. The α-N-carbamoyl protecting groupis removed from the resulting peptide-resin using a reagent such astrifluoroacetic acid or piperidine, and the coupling reaction repeatedwith the next desired N-protected amino acid to be added to the peptidechain. Suitable N-protecting groups are well known in the art, witht-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) beingpreferred herein.

The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.). The following side-chain protected aminoacids may be purchased from Applied Biosystems, Inc.:BSD-112344.1-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl),Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z),Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt),Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased fromApplied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole,dimethylsulfide, phenol, ethanedithiol, and thioanisole may be obtainedfrom Aldrich Chemical Company (Milwaukee, Wis.). Air Products andChemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic acid andmethanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).

Solid phase peptide synthesis may be carried out with an automaticpeptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry(see, Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resinsmay be cleaved with HF (−50° C. to 0° C., 1 hour). The peptide may beextracted from the resin with alternating water and acetic acid, and thefiltrates lyophilized. The Fmoc-peptide resins may be cleaved accordingto standard methods (Introduction to Cleavage Techniques, AppliedBiosystems, Inc., 1990, pp. 6-12). Peptides may also be assembled usingan Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).

Peptides may be purified by RP-HPLC (preparative and analytical) using aWaters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ,2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, andpurity may be determined using a C4, C8 or C18 analytical column (5μ,0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) maybe delivered to the analytical column at a flowrate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen, et al., The Pico Tag Method: A Manual of AdvancedTechniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation,Milford, Ma. (1989)). Fast atom bombardment analysis may be carried outby M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.Electrospray mass spectroscopy may be carried and on a VG-Trio machine.

Exendins and exendin agonists that are peptides may also be preparedusing recombinant DNA techniques, using methods now known in the art.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2dEd., Cold Spring Harbor (1989). Alternatively, such compounds may beprepared by homogeneous phase peptide synthesis methods. Non-peptidecompounds useful in the present invention may be prepared by art-knownmethods. For example, phosphate-containing amino acids and peptidescontaining such amino acids, may be prepared using methods known in theart. See, e.g., Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).

Methods of the Invention

The methods and compositions of this invention may be used to treatPCOS. Many of the symptoms associated with PCOS stem from an underlyinginsulin resistance. The symptoms associated with PCOS include insulinresistance, hyperinsulinemia, hyperandrogenism, type-2 diabetesmellitus, irregular menses, anovulation and infertility. Therefore, thepresent invention provides methods of treating insulin resistance in asubject suffering from PCOS comprising the step of administering GLP-1.

Insulin resistance may be due to any one or more events includingabnormal prereceptor (e.g., abnormal ligand or competition), receptor(e.g., abnormal structure, affinity of ligand to receptor, or number ofreceptors), or postreceptor (e.g., abnormal signaling) events. Insulinresistance may be determined by a number of methods known in the art.For example, the euglycemic hyperinsulinemic clamp technique may be usedto diagnose insulin resistance (Rao, G., Am. Fam. Physician (2001)63:1159-63). This technique involves intravenous administration of aninsulin dose while simultaneously maintaining glucose at a pre-set levelwithin the normal range by also administering glucose. At equilibrium,the amount of glucose uptake by a particular tissue in the presence of acertain dose of insulin can be calculated. Other methods used to detectinsulin resistance include the insulin suppression test, intravenousglucose tolerance test, and constant infusion of glucose with modelassessment (Rao, G., supra).

In another embodiment, the invention provides a method of preventing theonset of type-2 diabetes mellitus in a subject suffering from PCOScomprising the step of administering GLP-1, exendin, or agonists oranalogs of these compounds. Type-2 diabetes mellitus is often a resultof hyperinsulinemia caused by insulin resistance. Thus, treating insulinresistance in these patients would prevent the development of type-2diabetes mellitus. Methods of diagnosing type-2 diabetes mellitus arewell-known in the art.

In yet another embodiment, this invention provides a method of restoringregular menses, restoring regular ovulation and/or restoring fertilityin a subject suffering from PCOS comprising the step of administeringGLP-1, exendin, or agonists or analogs of these compounds. PCOS patientsoften exhibit hyperandrogenism, which is thought to be caused byhyperinsulinemia. The hyperandrogenism leads to follicular involution,anovulation and infertility. Thus, reducing insulin resistance byadministering GLP-1, exendin, or agonists or analogs of these compoundscan ameliorate hyperinsulinemia, thereby restoring regular menses,ovulation, and/or fertility.

In another embodiment, this invention provides a method for treatingPCOS comprising coadministering to a patient in need thereof GLP-1,exendin, or agonists or analogs of these compounds with a drug thatinduces ovulation (e.g., clomiphene, follistim, or Gonal-F).

In yet another embodiment, this invention provides a method for treatingPCOS comprising coadministering to a patient in need thereof GLP-1,exendin, or agonists or analogs of these compounds with ananti-androgenic drug, including but not limited to a birth control pill(e.g., progestogens and estrogens), spironolactone, flutamide andfinasteride.

In yet another embodiment, this invention provides a method for treatingPCOS comprising coadministering to a patient in need thereof GLP-1,exendin, or agonists or analogs of these compounds with aninsulin-sensitizing agent, including, but not limited to, metformin,D-Chiro-inositol, diazoxide, and PPAR inhibitors (e.g., troglitazone(Rezulin), rosiglitazone (Avandia) and pioglitazone (Actos)).

In another embodiment, this invention provides a method for treatingPCOS comprising coadministering to a patient in need thereof GLP-1,exendin, or agonists or analogs of these compounds with glucose. In amore preferred embodiment the glucose is administered intravenously.

When the compounds of this invention are administered in combinationtherapies as described above, they may be administered sequentially orconcurrently to the patient. Alternatively, the pharmaceuticalcompositions of this invention may be comprised of a combination of aGLP-1, exendin, or agonists or analogs of these compounds molecule andanother agent as described above.

In a preferred embodiment, the subject suffering from PCOS is a mammal,e.g., dog, cat, rodent. In a more preferred embodiment, the subjectsuffering from PCOS is a human.

Pharmaceutical Compositions

GLP-1, exendin, and agonists or analogs of these compounds may beformulated into pharmaceutical compositions for administration tosubjects, including humans. These pharmaceutical compositions,preferably include an amount of GLP-1, exendin, or agonists or analogsof these compounds effective to treat, e.g., insulin resistance, preventthe onset of type-2 diabetes mellitus, restore regular menses and/orovulation and treat infertility in a subject suffering from PCOS, and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers useful in these pharmaceuticalcompositions include, e.g., ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The compositions of the present invention may be administeredparenterally, orally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered by an infusionpump or subcutaneous injection of a slow release formulation

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art, using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage forms,including emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered once a day or on an “as needed” basis.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, capsules,tablets, aqueous suspensions or solutions. In the case of tablets fororal use, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried cornstarch. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically. Topical application can be effected in a rectalsuppository formulation (see above) or in a suitable enema formulation.Topically transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzylalcohol, and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH-adjusted sterile saline, or,preferably, as solutions in isotonic, pH-adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of GLP-1, exendin, or agonists or analogs of these compoundsthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. The compositions can be formulated so that adosage of between 0.1-1000 pmoles/kg body weight/minute (whenadministered by infusion) of GLP-1, exendin, or agonists or analogs ofthese compounds is administered to a patient receiving thesecompositions. In some embodiments of the invention, the dosage is 1-10pmoles/kg body weight/minute (when administered by infusion). In apreferred embodiment the dosage is 0.5-2.0 pmoles/kg/min whenadministered by intravenous infusion. The composition may beadministered as a single dose, multiple doses, or over an establishedperiod of time in an infusion.

In a preferred embodiment, GLP-1, exendin, or agonists or analogs ofthese compounds is administered to patients with confirmed polycysticovary syndrome. In another preferred embodiment, GLP-1, exendin, oragonists or analogs of these compounds is administered by injection atleast once a day or by continuous infusion via pump. In yet anotherpreferred embodiment, GLP-1, exendin, or agonists or analogs of thesecompounds is formulated for administration from a subcutaneous depotover a period of days to weeks, oral administration or by intermittentinhalation.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular GLP-1,exendin, or agonists or analogs of these compounds the patient's age,body weight, general health, gender, and diet, and the time ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease being treated. Judgment of such factors bymedical caregivers is within ordinary skill in the art. The amount ofGLP-1, exendin, or agonists or analogs of these compounds will alsodepend on the individual patient to be treated, the route ofadministration, the type of formulation, the characteristics of thecompound used, the severity of the disease, and the desired effect. Theamounts of GLP-1, exendin, or agonists or analogs of these compounds canbe determined by pharmacological and pharmacokinetic principleswell-known in the art.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES

1. Treatment of a PCOS Patient with GLP-1

Patients with PCOS are exclusively women. Typically, premenopausal womenmanifest the disease with complaints of irregular menses, infertility,excessive growth of body hair, acne and loss of scalp hair.Postmenopausal women may have all of these complaints except irregularmenses. Obesity, hypertension and diabetes are disorders that commonlyaccompany PCOS.

The diagnosis of PCOS will be confirmed by measuring the level of serumtestosterone and/or the ratio of serum LH/FSH. Elevated levels of serumtestosterone (>60 ng/ml) or an abnormal serum LH/FSH ratio (<2.5) areindicative of PCOS.

Patients with confirmed PCOS will be treated with GLP-1. GLP-1 will beadministered by injection once or more each day or by continuousinfusion via pump, which delivers a steady amount of drug.Alternatively, GLP-1 will be formulated for administration from asubcutaneous depot over days to weeks, by intermittent inhalation ororally.

Irrespective of the mode of administration, the total amount of GLP-1delivered into the blood of a patient with PCOS will be in the range of720 to 2880 picomoles/kg/day. This is equivalent to 0.5-2.0pmoles/kg/min when administered by intravenous infusion.

The efficacy of GLP-1 will be established by determining theamelioration or reversal of the presenting complaint, including but notlimited to normalization of menses, restoration of fertility, loss ofexcess body hair, resolution of acne and cessation of hair loss. Otherindicators of GLP-1 efficacy may be used including but not limited toserum testosterone levels and LH/FSH ratios. GLP-1 efficacy will bedetermined by a decrease in serum testosterone levels and an increase inthe LH/FSH ratio.

1. A method of treating a subject exhibiting at least one symptom of polycystic ovary syndrome (PCOS), said method comprising administering to said subject an amount effective to treat said at least one symptom of PCOS of a peptide compound capable of binding to and activating a GLP-1 receptor in vivo, wherein said peptide comprises an exendin analog, and wherein said exendin analog has a sequence selected from the group consisting of SEQ ID NOS: 20, 22, 23, 24, 25, 26, 27 and any combination thereof.
 2. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 20. 3. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 22. 4. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 23. 5. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 24. 6. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 25. 7. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 26. 8. The method of claim 1, wherein said exendin analog comprises SEQ ID NO:
 27. 9. The method of claim 1, wherein administration of said peptide compound reduces insulin resistance in said subject.
 10. The method of claim 1, wherein administration of said peptide compound prevents the onset of type-2 diabetes in said subject.
 11. The method of claim 1, wherein administration of said peptide compound restores regular menses in said subject.
 12. The method of claim 1, wherein administration of said peptide compound restores regular ovulation in said subject.
 13. The method of claim 1, wherein administration of said peptide compound restores fertility in said subject.
 14. The method of claim 1, wherein administration of said peptide compound prevents spontaneous abortion in said subject. 